Monday, 20 April 2015

ASSAY FOR BIDIRECTIONAL REPLICATION OF DNA: Prokaryotic replication pattern (Theta replication), autoradiograph for theta replication, radio thymidine experiment, eukaryotic replication pattern (pulse method).

In the early 1960s, John Cairns labeled replicating E. coli DNA with a radioactive DNA precursor, then subjected the labeled DNA to autoradiography. Figure shows the results, along with Cairns’s interpretation. The structure represented in Figure is a so-called theta structure because of its resemblance to the Greek letter u (theta).  This drawing shows that DNA replication begins with the creation of a “bubble”—a small region where the parental strands have separated and progeny DNA has been synthesized. As the bubble expands, the replicating DNA begins to take on the theta shape.

The u structure contains two replicating forks, marked X and Y in Figure. This raises an important question: Does one of these forks, or do both, represent sites of active DNA replication? In other words, is DNA replication  unidirectional, with one fork moving away from the other, which remains fixed at the origin of replication? Or is it bidirectional, with two replicating forks moving in opposite directions away from the origin? Cairns’s autoradiographs were not designed to answer this question, but a subsequent study on Bacillus subtilis replication performed by Elizabeth Gyurasits and R.B. Wake showed clearly that DNA replication in that bacterium is bidirectional.

These investigators’ strategy was to allow B. subtilis cells to grow for a short time in the presence of a weakly radioactive DNA precursor, then for a short time with a more strongly radioactive precursor. The labeled precursor was the same in both cases: [3H]thymidine. Tritium (3H) is especially useful for this type of autoradiography because its radioactive emissions are so weak that they do not travel far from their point of origin before they stop in the photographic emulsion and create silver grains. This means that the pattern of silver grains in the autoradiograph will bear a close relationship to the shape of the radioactive DNA. It is important to note that unlabeled DNA does not show up in the autoradiograph. The pulses of label in this experiment were short enough that only the replicating bubbles are visible. You should not mistake these for whole bacterial chromosomes such as in Figure. If you look carefully at Figure, you will notice that the pattern of silver grains is not uniform. They are concentrated near both forks in the bubble. This extra labeling identifies the regions of DNA that were replicating during the “hot,” or high-radioactivity, pulse period. Both forks incorporated extra label, showing that they were both active during the hot pulse. Therefore, DNA replication in B. subtilis is bidirectional; two forks arise at a fixed starting point—the origin of replication—and move in opposite directions around the circle until they meet on the other side. Later experiments employing this and other techniques have shown that the E. coli chromosome also replicates bidirectionally.

J. Huberman and A. Tsai have performed the same kind of autoradiography experiments in a eukaryote, the fruit fl y Drosophila melanogaster. Here, the experimenters gave a pulse of strongly radioactive (high specific activity) DNA precursor, followed by a pulse of weakly radioactive (low specific activity) precursor. Alternatively, they reversed the procedure and gave the low specific activity label first, followed by the high. Then they autoradiographed the labeled insect DNA. The spreading of DNA in these experiments did not allow the replicating bubbles to remain open; instead, they collapsed and appear on the autoradiographs as simple streaks of silver grains. One end of a streak marks where labeling began; the other shows where it ended. But the point of this experiment is that the streaks always appear in pairs. The pairs of streaks represent the two replicating forks that have moved apart from a common starting point. Why doesn’t the labeling start in the middle, at the origin of  replication, the way it did in the experiment with B. subtilis DNA? In the B. subtilis experiment, the investigators were able to synchronize their cells by allowing them to germinate from spores, all starting at the same time. That way they could get label into the cells before any of them had started making DNA (i.e., before germination). Such synchronization was not tried in the Drosophila experiments, where it would have been much more difficult. As a result, replication usually began before the label was added, so a blank area arises in the middle where replication was occurring but no label could be incorporated.

Notice the shape of the pairs of streaks in Figure. They taper to a point, moving outward, rather like an old fashioned waxed mustache. That means the DNA incorporated highly radioactive label first, then more weakly radioactive label, leading to a tapering off of radioactivity moving outward in both directions from the origin of replication. The opposite experiment—“cooler” label first, followed by “hotter” label—would give a reverse mustache, with points on the inside. It is possible, of course, that closely spaced, independent origins of replication gave rise to these pairs of streaks. But we would not expect that such origins would always give replication in opposite directions. Surely some would lead to replication in the same direction, producing asymmetric autoradiographs such as the hypothetical one in Figure. But these were not seen. Thus, these autoradiography experiments confirm that each pair of streaks we see really represents one origin of replication, rather than two that are close together. It therefore appears that replication of Drosophila DNA is bidirectional.

The theta mode of DNA replication in Escherichia coli. (a) An autoradiograph of replicating E. coli DNA with an interpretive diagram. The DNA was allowed to replicate for one whole generation and part of a second in the presence of radioactive nucleotides to label the DNA. The interpretive diagram to the right uses red to represent labeled DNA and blue to represent unlabeled parental DNA. (b) Detailed description of the theta mode of DNA replication.

Experimental demonstration of bidirectional DNA replication. 

Autoradiograph of replicating Bacillus subtilis DNA. Dormant bacterial spores were germinated in the presence of lowradioactivity DNA precursor, so the newly formed replicating bubbles immediately became slightly labeled. After the bubbles had grown somewhat, a more radioactive DNA precursor was added to label the DNA for a short period.

Experimental demonstration of bidirectional DNA replication. 

 Interpretation of the autoradiograph. The purple color represents the slightly labeled DNA strands produced during the low-radioactivity pulse. The orange color represents the more highly labeled DNA strands produced during the later, high-radioactivity pulse. Because both forks picked up the highradioactivity label, both must have been functioning during the high-radioactivity pulse. DNA replication in B. subtilis is therefore bidirectional.

Bidirectional DNA replication in eukaryotes. 

Autoradiograph of replicating Drosophila melanogaster DNA, pulselabeled first with high-radioactivity DNA precursor, then with low. Note the pairs of streaks (denoted by brackets) tapering away from the middle. This reflects the pattern of labeling of a replicon with a central origin and two replicating forks.

 Idealized diagram showing the patterns observed with high-, then low-radioactivity labeling, and the pattern expected if the pairs of streaks represent two independent unidirectional replicons whose replicating forks move in the same direction. The latter pattern was not observed.

 Autoradiograph of replicating embryonic Triturus vulgaris DNA. Note the constant size and shape of the pairs of streaks, suggesting that all the corresponding replicons began replicating at the same time.


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